Structure-Based Virtual Screening Protocol for in Silico Identification of

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A Structure-based Virtual Screening Protocol for in silico Identification of Potential Thyroid Disrupting Chemicals Targeting Transthyretin Jin Zhang, Afshan Begum, Kristoffer Brännström, Christin Grundström, Irina Iakovleva, Anders Olofsson, Astrid Elisabeth Sauer-Eriksson, and Patrik L. Andersson Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b02771 • Publication Date (Web): 26 Sep 2016 Downloaded from http://pubs.acs.org on September 28, 2016

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Environmental Science & Technology

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A Structure-based Virtual Screening Protocol for in

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silico Identification of Potential Thyroid Disrupting

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Chemicals Targeting Transthyretin

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Jin Zhang†, Afshan Begum†, Kristoffer Brännström‡, Christin Grundström†, Irina Iakovleva‡,

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Anders Olofsson‡, A. Elisabeth Sauer-Eriksson†, Patrik L. Andersson†*

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†Department of Chemistry, Umeå University, SE-901 87 Umeå, Sweden

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‡Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå,

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Sweden

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Corresponding Author

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*Email: [email protected]. Phone: +46-90-786-5266, Fax: +46-90-786-7655

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ACS Paragon Plus Environment

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ABSTRACT

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Thyroid disruption by xenobiotics is associated with a broad spectrum of severe adverse

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outcomes. One possible molecular target of thyroid hormone disrupting chemicals (THDCs) is

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transthyretin (TTR), a thyroid hormone transporter in vertebrates. To better understand the

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interactions between TTR and THDCs, we determined the crystallographic structures of human

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TTR in complex with perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA), and

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2,2',4,4'-tetrahydroxybenzophenone (BP2). The molecular interactions between the ligands and

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TTR were further characterized using molecular dynamics simulations. A structure-based virtual

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screening (VS) protocol was developed with the intention of providing an efficient tool for the

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discovery of novel TTR-binders from the Tox21 inventory. Among the 192 predicted binders,

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twelve representatives were selected and their TTR binding affinities were studied with

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isothermal titration calorimetry, of which seven compounds had binding affinities between 0.26-

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100 µM. To elucidate structural details in their binding to TTR, crystal structures were

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determined of TTR in complex with four of the identified compounds including 2,6-dinitro-p-

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cresol, bisphenol S, clonixin and triclopyr. The compounds were found to bind in the TTR

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hormone binding sites as predicted. Our results show that the developed VS protocol is able to

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successfully identify potential THDCs and we suggest that it can be used to propose THDCs for

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future toxicological evaluations.

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INTRODUCTION

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Thyroid hormone disrupting chemicals (THDCs) are anthropogenic compounds that disrupt the

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homeostasis of the thyroid hormone (TH) system1 and can induce disorders in physiological

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processes, including macronutrient metabolism, energy balance, brain development, and

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reproduction.2 Exposure to THDCs poses a significant threat to human health, especially during

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fetal development. Prenatal hypothyroxinemia induced by THDCs can impair the development

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of the embryonic brain and lead to neurologic deficits such as visuo-spatial processing

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difficulties3 and reduced learning and memory.4

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Transthyretin (TTR), a homotetrameric serum protein that transports L-thyroxine (T4),5 has

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been suggested to be a possible molecular target of THDCs such as per and polyfluoroalkyl

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substances (PFASs)6 and hydroxylated polychlorinated biphenyls (OH-PCBs).7 TTR is the

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primary TH transporter in developing rodents,8 fish, birds, and amphibians9 and THDCs can

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disrupt TH transport by competitively binding to the two identical thyroxine binding sites (TBSs)

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situated at the dimeric interface of TTR.10,11 TTR can also mediate delivery of THDCs across the

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placental and blood-brain barrier into the fetus and brain.12-14 Most TTR in human plasma resides

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in the apo form and the concentration of tetrameric TTR in plasma (4-7 µM)15,16 is significantly

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higher than that of T4 (0.112 µM)17. This means that low affinity TTR-binders can, without

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competing with T4, bind to TTR and be transported to vital organs where they may subsequently

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cause adverse health effects including developmental disorders.18,19 Inuit populations that are

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chronically exposed to relatively high levels of TTR-binding contaminants showed normal T4

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levels, but TTR-assisted accumulation of THDCs could potentially cause developmental

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disorders.20-22 It is thus critical to understand the molecular interactions between THDCs and

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TTR, and more importantly to identify and reduce the exposure to THDCs that target TTR.


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Virtual screening (VS) using molecular docking is an efficient means to reduce costs and

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animal testing by identifying the most hazardous compounds in risk assessment processes or

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prioritizing the most promising candidates in drug discovery.23,24 The method has been used to

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propose novel TTR amyloid inhibitors25 and to identify environmental pollutants targeting

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estrogen receptor.26,27 Molecular docking can give insights into ligand-protein binding

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conformations, and this was used to reveal interactions between PFASs and human liver fatty-

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acid binding proteins.28 Such studies provide a better understanding of critical molecular

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interactions between hazardous compounds and their targets.

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Crystal structures of ligand-protein complexes are an essential component in the development

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of VS protocols. Over 200 crystal structures of TTR have been reported in the Protein Data Bank

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(PDB),29,30 but only a few of these are in complex with environmental pollutants or their

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metabolites. These include TTR complexes with pentabromophenol31 and OH-PCBs.10 In

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addition, we recently determined the crystal structure of TTR with the brominated flame

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retardant tetrabromobisphenol A (TBBPA).32 Additional crystal structures of TTR complexes

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with environmental pollutants with large chemical variation would improve the development of a

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VS protocol for environmental pollutants and increase our understanding of their interactions

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with TTR at the molecular level.

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In this study, we determined the X-ray structures of human TTR in complex with three

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emerging contaminants that were shown to bind to TTR: perfluorooctanoic acid (PFOA),

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perfluorooctanesulfonic acid (PFOS), and 2,2’,4,4’-tetrahydroxybenzophenone (BP2).6,33 PFOA

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and PFOS are commonly used as stain, water, and grease repellents in food packaging, carpets,

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and textiles as well as in fire-fighting foams.34 BP2 is used as a UV absorber in personal care

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products and plastic materials.35 Exposure to PFOS and PFOA is associated with hypothyroidism


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and an increasing incidence of osteoporosis.36-38 BP2 might cause the development of

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endometriosis.39 These ligands together with TBBPA cover a wide chemical variation of TTR

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ligands. Their TTR binding activities in human plasma were measured with a previously

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established plasma assay.32,40 The ligand-TTR interactions in the X-ray structures were further

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studied by performing molecular dynamics (MD) simulations, and residues important for the

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interactions were proposed by decomposition of ligand-binding free energies using the

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Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) method. Based on the novel

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X-ray structures and the molecular interaction information, a structure-based VS protocol was

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developed to identify THDCs targeting TTR from the Tox21 inventory of 7,849 organic

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compounds.41 Twelve representative industrial compounds were selected for in vitro studies

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using isothermal titration calorimetry (ITC). Of these, four hits have been co-crystallized with

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TTR to study their binding conformations.

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MATERIALS AND METHODS

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Crystallization of the TTR-THDC complexes. Human wild-type TTR was purified and

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crystallized in complex with PFOA, PFOS, BP2 and with four confirmed hits (bisphenol S

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(BPS), clonixin, 2,6-dinitro-p-cresol (DNPC), and triclopyr) following the procedures described

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previously.42,43 The puriﬁed TTR was dialyzed against 10 mM Na-phosphate buffer with 100

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mM KCl (pH 7.6) and concentrated to 5 mg/mL using an Amicon Ultra centrifugal ﬁlter device

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(Millipore, 3 kDa molecular-weight cutoff) and co-crystallized at room temperature with a 5-

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molar excess of the compounds using the vapor-diffusion hanging drop method. A drop

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containing 3 µL protein solution was mixed with 3 µL precipitant and equilibrated against 1 mL

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reservoir solution containing 1.3–1.6 M sodium citrate and 3.5% v/v glycerol at pH 5.5 in 24-


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well Linbro plates. Crystals grew to dimensions of 0.2 × 0.2 × 0.3 mm3 after 5 days. The crystals were cryoprotected with 12% v/v glycerol.

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Crystallographic data collection, integration, and structure determination. The X-ray

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diffraction data of the complexes were collected at the European Synchrotron Radiation Facility

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(Grenoble, France) on beamline ID29 or ID23-1 and in-house instruments (Table S1 and S2).

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The structure of human TTR (PDB ID: 1F415) and X-ray data from 38.2–2.5 Å resolution were

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used in molecular replacement searches with the program PHASER.44 The models were refined

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against all of the diffraction data using PHENIX.45 At the end of structure refinement,

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anisotropic B-factors were refined. Manual map inspection was performed with COOT.46 The

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details of the refinement statistics are shown in Table S1 and S2. Molecular graphics were

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produced using CCP4mg.47 Structure factors and coordinates of the TTR complexes have been

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deposited with the Protein Data Bank (PDB ID: 5JID (PFOA), 5JIM (PFOS), 5JIQ (BP2), 5LAF

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(DNPC), 5L4I (clonixin), 5LAJ (BPS) and 5L4M (triclopyr)).

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Molecular dynamics simulations. The MD simulations were prepared based on the three

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structures of TTR complexes with BP2, PFOA (sharing similar binding profiles with PFOS), and

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TBBPA32. Each system was prepared following the procedures described in the supporting

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information. The MD simulations of each prepared system were performed using Amber 1448

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with the following steps: 1) each system was minimized with two-stage energy minimizations, 2)

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each system was gradually heated to 310 K and then equilibrated under NPT conditions for 1 ns,

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and 3) the 20 ns production MD was performed under NPT conditions using a Langevin

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thermostat and Berendsen barostat. Details of the simulation are described in the supporting

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information.


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MM-GBSA free-energy decomposition. We calculated the binding free energies of BP2,

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PFOA, and TBBPA to TTR using the MM-GBSA method in AmberTools 1548 based on the last

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10 ns of the MD trajectory. The contributions of each residue in the TBS to the ligand binding

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were determined by the pairwise decomposition method. The five residues that contributed the

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most to the enthalpic binding free energy of each ligand were considered to be important for

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ligand-TTR interactions (Table S3). The entropy contributions were neglected due to low

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prediction accuracy.49 The procedures and parameters are described in the supporting

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information.

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Collection and preparation of the ligand dataset. Two ligand sets were used in the study –

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the TTR benchmarking set and the Tox21 inventory.41 The TTR benchmarking set (Table S4)

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contained 155 TTR-binders and 10,197 in silico generated decoys (inactives).50 The TTR-binders

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were collected from the scientific literature using an activity cutoff of Ki or IC50 ≤ 100 µM as

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suggested by the US EPA in the studies of THDCs51 and estrogen disruptors26,52 and used in the

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Tox21Challenge in silico model-developing exercise (more explanations given in supporting

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information).53 The benchmarking set was used in the development and evaluation of the VS

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protocol. The Tox21 dataset contains 7,849 industrial compounds and pharmaceuticals.54 For

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each compound in the two sets, a maximum of 32 low-energy conformations were generated by

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considering all chiral atoms using the Schrödinger LigPrep module under the OPLS_2005 force

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field,55 and their ionization and tautomeric states were determined at pH 7.0 ± 1.0.

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Molecular docking. Molecular docking models were developed using the TTR complex

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structures with BP2, PFOA, and TBBPA. The TBS situated at the BB’ interface was described

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by a grid box centered on its co-ligand. The prepared compounds in the TTR benchmarking set

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were docked into each structure using the Schrödinger Glide module under standard precision.56


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For the docking results, we assessed their VS performance and enrichment. The VS

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performance of each model was described with the area under curve (AUC) value of the receiver

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operating characteristic (ROC) curves. The AUC value was used because it is insensitive to the

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ratio of actives versus decoys in the benchmarking set.57 The enrichment was characterized by

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two enrichment factors (EFs) that were evaluated at 10% and 20% of the ranked database,

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referred as EF10% and EF20%, respectively. The AUC values and EFs are given in Table S5.

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Ligand-TTR interaction analysis. Residues important for ligand-TTR interactions were

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identified using the MM-GBSA binding free-energy decomposition. The W188 water molecule

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in the TTR-TBBPA complex was also considered because it was suggested to be critical for

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mediating hydrogen bonds (H-bonds) between TBBPA and Ser117.32 For each initial hit

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identified by molecular docking, we counted the number of electrostatic interactions (H-bonds

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and salt bridges) with polar residues (including W188) and hydrophobic interactions with non-

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polar residues based on their docking poses. The number of interactions together with the

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docking score was used to refine the initial hits and identify the bioactive compounds from the

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Tox21 inventory. The refined hits and their interactions with TTR are shown in Table S6. The

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known TTR-binders identified among the refined hits are given in Table S7.

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Measuring compound binding activity in human plasma. Binding activity of BP2, PFOS,

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and PFOA to TTR in the presence of human plasma was measured with a previously established

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plasma assay.40 Details of experimental procedures are given in supporting information.

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Isothermal Titration Calorimetry. The binding affinities of twelve selected potential TTR-

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binders were measured using an Auto-iTC200 (MicroCal, Malvern, UK) at 25 °C. The chemical

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information and properties of the twelve compounds are shown in Table S8. Stock solutions

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were prepared in dimethyl sulfoxide (DMSO) and diluted with phosphate-buffered saline (PBS,


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pH=7.4) to a final level of 5% DMSO immediately before the ITC experiments.40 Aliquots of

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each compound were titrated into the TTR buffer solution (30 or 90 µM) to reach a 10-fold molar

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excess. For the control experiment, the compounds were titrated into the cell with only buffer.

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Raw data were collected, subtracted for compound heats of dilution, and processed using the

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MicroCal Origin software. Calorimetric data (Table S9) were plotted and fitted using the

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standard single-site binding model to yield the binding affinities (Kd), enthalpy changes, and

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entropy changes.

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RESULTS AND DISCUSSION

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Crystal structures of the TTR-THDC complexes. We co-crystallized human wild-type TTR

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in complex with PFOA, PFOS, and BP2 and determined their structures at 1.2–1.4 Å resolution

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(Table S1). The structures showed that all three ligands bind at the deep end of the TBS (Figure

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1). Compared with T4, the three ligands have the ability to bind deeper in the pocket due to their

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smaller molecular size (Figure S1).

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Of particular interest are the TTR complex structures with PFOA and PFOS. The structures

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share three unique features. 1) The two ligands bind with their hydrophobic tail inside the pocket

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and their hydrophilic acid groups are surface exposed (Figure S2), which is different from our

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previous assumption.33 2) The hydrophobic tail bends at the second to last carbon atom of the

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ligand to accommodate the volume between two Ser117 residues and to allow the acid groups to

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form salt bridges with Lys15. 3) The bent hydrophobic tail forces the hydroxyl groups on Ser117

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to rotate away from the ligands (Figure 1), which creates a more hydrophobic pocket that favors

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the ligand-TTR interactions.

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BP2 interacts with TTR similarly as luteolin in the deep end of the TBS (Figure S2).58 Two

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hydroxyl groups form hydrogen bonds with Ser117 residues, and the carbonyl group interacts


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with Thr119. Interactions between the aromatic ring of BP2 and TTR involve van der Waal

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contacts with the side chain of Leu17.

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MD simulations for ligand-TTR interaction studies. We investigated the molecular

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interactions between TTR and THDCs by performing MD simulations on the TTR complex

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structures with BP2, PFOA, and TBBPA.32 All three ligands remained bound in the TBS, and the

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root-mean-squared-deviation (RMSD) of the protein and ligands was under 2 Å throughout the

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simulations (Figure S3 and Figure S4).

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The ligand-TTR binding free energies were calculated using the MM-GBSA method based on

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the last 10 ns of the MD trajectory. The calculated energies of the ligands correlate well with

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their previously measured potencies (Figure S5).6,33,59 The energies were further decomposed to

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reveal five residues of significance for the ligand-TTR interactions in each of the three complex

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structures (Table S3). Two types of ligand-TTR interactions were identified electrostatic

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interactions with the polar residues B-Thr119, B-Ser117, B’-Ser117, B-Lys15, and B’-Lys15 and

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hydrophobic interactions with the non-polar residues B-Leu110, B-Leu17, B’-Leu17, and B-

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Ala108. The electrostatic interactions account for specific ligand-TTR recognition and a large

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proportion of the binding free energies,60-62 whereas the hydrophobic interactions were less

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significant for ligand binding. The results also revealed that the three ligands interact with the

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residues deeper in the TBS, e.g. Ser117, whereas T4 interacts with residues at the opening of the

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TBS, e.g. Glu54.63 Information on the molecular interactions with the identified residues (Table

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S3) was used to better understand the ligand-TTR interaction and to refine potential TTR-binders

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for better identifying of bioactive compounds.

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Molecular docking model. The accuracy of the molecular docking using Glide was assessed

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by comparing the docking conformations of BP2, PFOA, and TBBPA with their X-ray


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structures.33 The RMSD values for the co-ligands were lower than 2 Å (Table S5), indicating that

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the molecular docking was able to reproduce the actual binding conformation. The prepared

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compounds in the benchmarking set were docked into each TTR complex, and the docking

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results were compared in terms of AUC values and enrichment factors (Table S5). The TTR-

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TBBPA structure showed the best results with AUC of 0.75 and good early enrichment (EF10%

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= 3.76 and EF20% = 2.95), and 141 binders were identified among the 155 binders in the

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benchmark set (Table S5). The docking models based on the TTR-PFOA and TTR-BP2

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structures had lower performances in identifying TTR-binders, probably because TBBPA best

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reflects the majority of the halogenated and hydroxylated aromatic compounds in the benchmark

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set. However, the two structures provide useful information for molecular interaction studies,

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and the TTR-PFOA structure reveals the correct binding orientations of PFASs in the TBS where

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the acidic group was found to interact with Lys15 rather than Ser117.

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The impact of water in the TBS of the TTR-TBBPA complex was studied by evaluating the

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model performance of the structure with and without water molecules in the TBS. The structure

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with water in the TBS outperformed the one without (Table S5 and Figure S6). After examining

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the ligand-protein interactions in the TTR-TBBPA structure, the W188 water molecule was

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found to be critical for mediating hydrogen bonds between the ligand and the Ser117 residues,

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which agrees well with previously reported data.32

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Binding activity of the studied THDCs to TTR in human plasma. We compared the

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binding activity of BP2, PFOA, and PFOS to TTR in human plasma in terms of their inhibitory

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concentrations at 50% (IC50) to prevent tetrameric dissociation in the established assay.32,40 BP2

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bound strongly to TTR with an IC50 of 4 µM, while PFOA (IC50 = 175 µM) and PFOS (IC50 =

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103 µM) had poor binding activity to TTR in plasma (Figure S7). Their differences in binding


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activity could be caused by the hydrophobic tails of the PFASs giving limited electrostatic

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contributions to their TTR binding, which are critical for molecular recognition.40,62 TBBPA has

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also shown strong binding to TTR (IC50 = 3.3 µM).32 Clearly, BP2 share characteristics with

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TBBPA in terms of molecular interactions with TTR. It is thus of interest to identify

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contaminants sharing similar molecular interactions with TTR as found in BP2 and TBBPA.

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Virtual screening of industrial compounds. The docking model developed using the TTR-

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TBBPA structure was used as the basis for the VS of the Tox21 inventory because of its high VS

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performance and the strong binding of TBBPA to TTR in human plasma. We applied a two-step

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procedure to identify potential TTR-binders from the Tox21 inventory. In the first step,

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compounds were docked into the TTR-TBBPA structure followed by ranking based on their

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standard precision docking score. For each industrial compound, all protonation states in the

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range of pH 7.0±1.0 were considered during the molecular docking. As suggested by previous

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studies,64,65 we selected its top scored protonation form (Table S4 and S6), since docking score

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considers both binding affinity and tautomeric ratio.66,67 The top 20% scored compounds

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covering 1,282 hits were considered as initial hits for TTR, and these were extracted for further

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analysis. In the second step, the initial hits were further refined by selecting those that showed at

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least three electrostatic interactions with the identified polar residues (including W188), and two

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hydrophobic interactions with the non-polar residues. The criteria were based on the interactions

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between TTR and TBBPA that binds to TTR with a Kd of 20 nM.32 The electrostatic interactions

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have been suggested to be critical for specific recognition of TTR.60-62 A total of 192 substances

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among the top 20% scored compounds also fulfilled the molecular interaction criteria (Table S6)

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and thus were predicted to be potential TTR binders by the VS protocol. Eleven known TTR-

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binders were identified (Table S7), including five pharmaceuticals (including diflunisal and


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aceclofenac),40 two flame retardants (TBBPA and tetrachlorobisphenol A), one herbicide (2,4,5-

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trichlorophenoxyacetic acid (2,4,5-T)),33 one UV filter BP2, and one T4 derivative (3,3´,5,5´-

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tetraiodothyroacetic acid).68 The recognition of these known TTR binders by the VS protocol

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indicates that it is capable of identifying potential TTR-binders for further toxicological

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investigations.

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Binding affinities of selected compounds. We selected twelve representative compounds

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(Table 1) for in vitro validation from the VS results based on their various applications, structural

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diversity, and environmental relevance. These compounds mainly represent herbicides and

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polymer additives (plasticizers and polymerization inhibitors) and include primarily halogenated

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and/or aromatic substances as well as nitro compounds. The thermodynamic profiles of the

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ligand-TTR binding were investigated using ITC (Figure 2). One TTR tetramer can bind two

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ligands. However, we fitted ITC thermograms (Figure 2) using a one-site binding model as

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binding of the first ligand dominates the total binding energy and its affinity is approximately

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100 times stronger than the second ligand.69,70 The ITC results showed that seven of the twelve

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compounds bound to TTR with Kd values below 100 µM (Table 1) indicating that our VS

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protocol gave a reasonable hit rate of approximately 60%, which can be compared with

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commonly practiced VS campaigns that generally have a hit rate of 20-40%.71,72 The

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thermodynamic data (Table S9) revealed that the binding of BPS, clonixin, fluroxypyr,

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mesotrione, and triclopyr was largely driven by the enthalpic component, whereas the binding

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energy of DNPC and picloram was mainly entropy driven. The affinities of the active

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compounds were not correlated with their docking scores (Figure S8), which was not surprising

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since docking scores generally give poor estimations of the binding affinities.73,74 However, the

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trend is correct (except DNPC), as more negative docking scores are associated with higher


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affinities. Notably, five predicted binders showed no activities in the ITC experiments (Figure

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S9) and we provide a thorough discussion on the potential reasons for their mispredictions in the

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supporting information.

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Crystal structures of the TTR-confirmed hits complexes. Four confirmed TTR-binders

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(clonixin, DNPC, triclopyr and BPS) were selected for co-crystallization with human wild-type

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TTR, considering their high binding affinities, diverse structures and various applications. The

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crystal structures were determined at 1.5-1.6 Å resolution (Table S2). Compared with T4, the

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four ligands bind deeper in the TBS due to their smaller molecular size (Figure 3). Clonixin

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interacts with TTR similarly as flufenamic acid.75 It forms H-bonds and salt bridges with Lys15

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(Figure S10). DNPC presents different binding conformations in the two TBSs. Furthermore, in

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the AA’ interface, two DNPC molecules bind into the TBS simultaneously where one DNPC

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interacts with Ser117 at the deep end of TBS, whereas the other DNPC binds to Lys15 at the

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cavity entrance (Figure S10). In the BB’ interface, DNPC also binds in two different

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orientations, however these conformations are different from the ones in AA’ and both cannot be

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occupied at the same time as their binding sites overlap (Figure 3). The multiple conformations

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of DNPC give better knowledge on interactions between TTR and nitro compounds, which

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provides guidance for molecular design and risk assessment. Triclopyr interacts with TTR by

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having electrostatic interactions with Lys15. BPS binds in the TBS similarly as TBBPA.32 Its

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hydroxyl groups interact with Lys15 and form water-bridged H-bonds with Thr119 and Ser117

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(Figure S8). We compared the docking pose of each ligand with its crystal conformation in the

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BB’ interface (Figure S11) and the results showed that we correctly predicted the binding

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conformations for clonixin, DNPC, and triclopyr. The inaccurate prediction of BPS may be due

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to its weaker affinity and high mobility in the TBS. Besides DNPC, EMD21388 was also


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reported to adapt to distinctive binding-modes in the two TBSs.76 Ensemble docking has been

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proposed as a suitable strategy for predicting conformation of such ligands,77,78 which however

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was not completed here due to lack of TTR structures with similar binding-modes to DNPC.

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Environmental implications of the potential THDCs. Seven compounds with diverse

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structures and various applications were identified as TTR-binders (Table 1). These compounds

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warrant further toxicological investigations considering the following two reasons: (1) humans

311

may be chronically exposed to the compounds at doses that are similar or significantly higher

312

than normal T4 level (0.112 µM)17. In vivo levels of the compounds have been reported to be

313

35844 µM (9.416 mg/ml) for clonixin79, 0.312 µM (0.08 µg/ml) for triclopyr80, 3.72 µM (0.93

314

µg/ml) for BPS81, and 9.19 µM (2.22 µg/ml) for picloram82. (2) Low-affinity binders could be

315

effectively transported by TTR and accumulated in vulnerable organs in vivo,12-14 since most

316

TTR in human plasma resides in the apo form.15-17 Their accumulations could subsequently

317

induce adverse effects including developmental disorders.18,19

318

Interestingly, BPS, a replacement for bisphenol A (BPA), bound to TTR with a Kd of 52 µM.

319

BPS is used extensively as a plasticizer in BPA-free consumer products and as an anticorrosive

320

agent in canned food. Despite claims that it is a safer alternative to BPA, BPS has been reported

321

to pose similar potential health hazards as BPA in a number of in vitro and in vivo studies.83

322

Embryonic exposure to BPS is associated with neurogenesis within the hypothalamus, causing

323

gestation and hyperactivity in juvenile zebrafish84 and non-associative learning impairments in

324

adult zebrafish.85 BPS can also decrease plasma TH levels and impair the reproductive potential

325

of zebrafish.86 It was also found in dust samples in twelve countries, and the highest median

326

concentration was in Greece (860 ng/g).87


15


Page 16 of 30

327

Clonixin was the strongest TTR binder identified in the Tox21 inventory and had a Kd of 0.26

328

µM. Clonixin is a non-steroidal anti-inflammatory drug, and it has a similar structure and TTR

329

binding affinity as meclofenamic acid.40 To our knowledge, its thyroid effects have not been

330

studied.

331

DNPC is used as an inhibitor of styrene polymerization and a dye intermediate, and this

332

molecule showed the second strongest interaction with TTR. DNPC is an uncoupler of oxidative

333

phosphorylation in humans, and it can cause acute hyperthermia, kidney and liver failure, and

334

cerebral edema.88 DNPC has a high potential for accumulation in biota and humans.88

335

The herbicides fluroxypyr, mesotrione, picloram, and triclopyr were identified as TTR-binders,

336

with triclopyr being the strongest binder. Triclopyr is used as an alternative to 2,4,5-T, and it has

337

been reported to cause thyroid-related neurotoxicity89 and developmental toxicity in rats.90 The

338

major metabolite of triclopyr is 3,5,6-trichloro-2-pyridinol,91 which is also a TTR-binder,33 and it

339

has been reported to disrupt the levels of T4 and thyroid stimulating hormone in humans.92

340

Exposure to triclopyr poses a significant threat to its applicators, and the amount in their urine

341

samples was reported to be 1–13 mg/day.93 The compound has been detected in water samples

342

from Australia,94 France,95 and multiple locations in the US.96,97 Environmental implications of

343

the other three herbicides are given in the supporting information.

344

In summary, we have determined the binding conformations of PFOS, PFOA, and BP2 in the

345

TBS of TTR. The structures of the complexes provide valuable information for improving our

346

understanding of the molecular interactions between TTR and emerging contaminants. PFOS and

347

PFOA were previously reported as strong TTR-binders, but our results indicate that they are

348

weak binders in plasma, whereas BP2 showed strong binding activity . BP2 is thus more likely to

349

be transported by TTR and accumulated in vulnerable organs. Based on the novel TTR complex


16

Page 17 of 30


350

structures and the information on molecular interactions obtained from MD simulations, a VS

351

protocol was developed for the identification of potential THDCs targeting TTR from the Tox21

352

inventory. The protocol proposed 192 industrial compounds as potential binders to TTR. Among

353

the twelve compounds tested using in vitro experiments, we identified seven novel TTR-binders

354

including clonixin, DNPC, triclopyr, fluroxypyr, BPS, picloram, and mesotrione. Most of these

355

have been detected in the environment and have shown adverse thyroid-related effects in animal

356

models. We further co-crystallized TTR with PBS, clonixin, DNPC, and triclopyr and their

357

structures showed that the compounds bind in the TBS as predicted by the VS protocol. We

358

suggest further in vivo studies on these TTR-targeting compounds especially as they may

359

accumulate in vulnerable organs.

360

Supporting Information

361

Details of the X-ray structures, plasma assay, and VS performance; the identified potential TTR-

362

binders and the compounds tested using ITC; procedures and results of the MD simulations and

363

MM-GBSA calculations can be found in the supporting information. This information is

364

available free of charge via the Internet at http://pubs.acs.org/.

365

ACKNOWLEDGMENT

366

This study was financed by the MiSSE project through grants from the Swedish Research

367

Council for the Environment, Agricultural Sciences and Spatial Planning (Formas) (210-2012-

368

131) and by the Swedish Research Council (VR) (521-2011-6427). The MD simulations were

369

conducted using the High Performance Computing Center North.

370


17


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(77) Totrov, M.; Abagyan, R., Flexible ligand docking to multiple receptor conformations: a practical alternative. Curr. Opin. Struct. Biol. 2008, 18, (2), 178-84. (78) Frimurer, T. M.; Peters, G. H.; Iversen, L. F.; Andersen, H. S.; Moller, N. P.; Olsen, O. H., Ligand-induced conformational changes: improved predictions of ligand binding conformations and affinities. Biophys. J. 2003, 84, (4), 2273-81. (79) Bica, A.; Farinha, A.; Blume, H.; Barbosa, C. M., Determination of clonixin in plasma and urine by reversed-phase high-performance liquid chromatography. J. Chromatogr. A 2000, 889, (1-2), 135-41. (80) Marin-Water-Agency, Vegetation Management Plan 2010. www.marinwater.org/DocumentCenter/ (accessed Aug 6,2016). (81) Liao, C.; Liu, F.; Alomirah, H.; Loi, V. D.; Mohd, M. A.; Moon, H. B.; Nakata, H.; Kannan, K., Bisphenol S in urine from the United States and seven Asian countries: occurrence and human exposures. Environ. Sci. Technol. 2012, 46, (12), 6860-6. (82) Hall, J. C.; Deschamps, R. J. A.; Krieg, K. K., Immunoassays for the Detection of 2,4-D and Picloram in River Water and Urine. J. Agric. Food Chem. 1989, 37, (4), 981-984. (83) Rochester, J. R.; Bolden, A. L., Bisphenol S and F: A Systematic Review and Comparison of the Hormonal Activity of Bisphenol A Substitutes. Environ. Health Perspect. 2015, 123, (7), 643-50. (84) Kinch, C. D.; Ibhazehiebo, K.; Jeong, J. H.; Habibi, H. R.; Kurrasch, D. M., Low-dose exposure to bisphenol A and replacement bisphenol S induces precocious hypothalamic neurogenesis in embryonic zebrafish. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, (5), 1475-80. (85) Mersha, M. D.; Patel, B. M.; Patel, D.; Richardson, B. N.; Dhillon, H. S., Effects of BPA and BPS exposure limited to early embryogenesis persist to impair non-associative learning in adults. Behav Brain Funct 2015, 11, 27. (86) Naderi, M.; Wong, M. Y.; Gholami, F., Developmental exposure of zebrafish (Danio rerio) to bisphenol-S impairs subsequent reproduction potential and hormonal balance in adults. Aquat. Toxicol. 2014, 148, 195-203. (87) Wang, W.; Abualnaja, K. O.; Asimakopoulos, A. G.; Covaci, A.; Gevao, B.; JohnsonRestrepo, B.; Kumosani, T. A.; Malarvannan, G.; Minh, T. B.; Moon, H. B.; Nakata, H.; Sinha, R. K.; Kannan, K., A comparative assessment of human exposure to tetrabromobisphenol A and eight bisphenols including bisphenol A via indoor dust ingestion in twelve countries. Environ. Int. 2015, 83, 183-91. (88) ATSDR Toxicological Profile for Dinitrocresols; USA Agency for Toxic Substances and Disease Registry (ATSDR), 1995. (89) Reddy, T. P.; Manczak, M.; Calkins, M. J.; Mao, P.; Reddy, A. P.; Shirendeb, U.; Park, B.; Reddy, P. H., Toxicity of neurons treated with herbicides and neuroprotection by mitochondriatargeted antioxidant SS31. Int. J. Environ. Res. Public Health 2011, 8, (1), 203-21. (90) Carney, E. W.; Billington, R.; Barlow, S. M., Developmental toxicity evaluation of triclopyr butoxyethyl ester and triclopyr triethylamine salt in the CD rat. Reprod. Toxicol. 2007, 23, (2), 165-74. (91) Schmidt, L.; Muller, J.; Goen, T., Simultaneous monitoring of seven phenolic metabolites of endocrine disrupting compounds (EDC) in human urine using gas chromatography with tandem mass spectrometry. Anal. Bioanal. Chem. 2013, 405, (6), 2019-29. (92) Fortenberry, G. Z.; Hu, H.; Turyk, M.; Barr, D. B.; Meeker, J. D., Association between urinary 3, 5, 6-trichloro-2-pyridinol, a metabolite of chlorpyrifos and chlorpyrifos-methyl, and serum T4 and TSH in NHANES 1999-2002. Sci. Total Environ. 2012, 424, 351-5.


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(93) Gosselin, N. H.; Brunet, R. C.; Carrier, G.; Dosso, A., Worker exposures to triclopyr: risk assessment through measurements in urine samples. Ann. Occup. Hyg. 2005, 49, (5), 415-22. (94) Tran, A. T.; Hyne, R. V.; Doble, P., Determination of commonly used polar herbicides in agricultural drainage waters in Australia by HPLC. Chemosphere 2007, 67, (5), 944-53. (95) Botta, F.; Fauchon, N.; Blanchoud, H.; Chevreuil, M.; Guery, B., Phyt'Eaux Cites: application and validation of a programme to reduce surface water contamination with urban pesticides. Chemosphere 2012, 86, (2), 166-76. (96) Battaglin, W. A.; Rice, K. C.; Focazio, M. J.; Salmons, S.; Barry, R. X., The occurrence of glyphosate, atrazine, and other pesticides in vernal pools and adjacent streams in Washington, DC, Maryland, Iowa, and Wyoming, 2005-2006. Environ. Monit. Assess. 2009, 155, (1-4), 281307. (97) Tagert, M. L.; Massey, J. H.; Shaw, D. R., Water quality survey of Mississippi's Upper Pearl River. Sci. Total Environ. 2014, 481, 564-73. (98) Purkey, H. E.; Dorrell, M. I.; Kelly, J. W., Evaluating the binding selectivity of transthyretin amyloid fibril inhibitors in blood plasma. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, (10), 5566-71.

655 656 657 658


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659 660 661


Graphical abstract

662 663 664


25


Page 26 of 30

665 666

Table 1. Studied compounds with abbreviations, chemical abstract services (CAS) registry

667

numbers, usage, and binding affinities (Kd) determined using isothermal titration calorimetry. Compounds

Abbreviations

CAS

Usage

Kd (µM)a

3,3',5,5'-tetraiodo-L-thyronine

T4

51-48-9

Positive Control

0.09b

2-nitro-5-(2-chloro-4Acifluorfen trifluoromethylphenoxy)benzoic acid

50594-66-6

Herbicide

ND

2,2'-dihydroxy-4,4'dimethoxybenzophenone

BP6

131-54-4

UV absorber

ND

4,4'-dihydroxydiphenyl sulfone

BPS

80-09-1

Plasticizer

52

2-(3-chloro-2methylanilino)pyridine-3carboxylic acid

Clonixin

17737-65-4

Pharmaceutic 0.26 al

2,6-dinitro-p-cresol

DNPC

609-93-8

Polymerizati on inhibitor

1.3

diphenolic acid

DPA

126-00-1

Plasticizer

ND

2-[(4-amino-3,5-dichloro-6fluoro-2-pyridinyl)oxy]acetic acid

Fluroxypyr

69377-81-7

Herbicide

45

L-γ-glutamyl-p-nitroanilide

GPNA

67953-08-6

Food additive

ND

2-[4-(methylsulfonyl)-2nitrobenzoyl]-1,3cyclohexanedione

Mesotrione

104206-82-8

Herbicide

99

3,5,6-trichloro-4-aminopicolinic Picloram acid

1918-02-1

Herbicide

63

1-(4-sulfophenyl)-3-carboxy-5pyrazolone

PyT

118-47-8

Dye

ND

3,5,6-trichloro-2pyridinyloxyacetic acid

Triclopyr

55335-06-3

Herbicide

4.6


26

Page 27 of 30

668 669 670


a

ND refers to non-detected affinity. bThe binding affinity of T4 is only an approximate value due to its thermal instability and poor solubility in the isothermal titration calorimetry buffer. The approximated binding affinity is similar to the value of 84 nM reported previously.98

671 672


27


Page 28 of 30

673

674 675

Figure 1. (A) The TTR monomers in the dimer structure are shown as ribbons and are labeled A

676

and B. The symmetry-related monomers are labeled A´ and B´. Locations of the thyroxine

677

binding sites (TBSs) are shown as gray surfaces. (B) Close-up view of the crystallographic

678

binding conformations of (a) BP2, (b) PFOA, and (c) PFOS in the TBS. The refined (2|Fo|−|Fc|)

679

electron density is shown in gray mesh at 1σ level, and the anomalous difference map showing

680

the position of the sulfur atoms in PFOS is shown in violet mesh at 3σ level. The water

681

molecules present around the ligands are shown as red spheres.

682


28

Page 29 of 30


683

684 685

Figure 2. Structures of active compounds and thermograms of their binding to TTR as

686

determined by isothermal titration calorimetry. The abbreviations of the compounds are given in

687

Table 1.

688


29


Page 30 of 30

689

690 691

Figure 3. Crystallographic binding conformations of the selected active compounds and T4 in

692

the thyroid-binding site of TTR; (a) clonixin, (b) DNPC (in the AA’ interface), (c) DNPC (in the

693

BB’ interface), (d) triclopyr, (e) T4 (PDB ID: 2ROX, for comparison), and (f) BPS. The refined

694

(2|Fo|−|Fc|) electron density is shown in gray mesh at 1σ level. The water molecules present

695

around the ligands are shown as red spheres. The electron density of the second ring of BPS was

696

poorly defined probably due to high mobility.

697


30

Structure-Based Virtual Screening Protocol for in Silico Identification of

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